Animal Cell Culture: Primary Cultures, Cell Lines, Methods, and Clinical Applications

Before animal cell culture existed, growing poliovirus in the laboratory meant injecting it into the spinal cord of living monkeys. It worked but it was slow, expensive, ethically problematic, and produced virus with uncontrollable variability. Jonas Salk's team needed to grow poliovirus at industrial scale to make a vaccine that could protect millions of children. The breakthrough came in the 1950s: growing the virus in cells taken from monkey kidney tissue and maintained in glass flasks. The polio vaccine followed directly from that capability and so did the modern field of vaccine production.

Today the same fundamental technique underpins nearly everything in clinical and research virology: isolating an unknown virus from a patient specimen, producing vaccines for rabies, hepatitis B, measles, and chickenpox, testing anticancer drugs, studying how pathogens interact with human cells, and manufacturing recombinant proteins. The cells have replaced the animal.

This article covers the foundational concepts behind animal cell culture: the types of cells used (primary versus cell lines, finite versus continuous), the methods for establishing and maintaining cultures, and the clinical and research applications including the one important thing continuous cell lines cannot do despite their many advantages that make this technique still central to medical microbiology.

Examples of cells used to culture are fibroblast, lymphocytes, cells from cardiac and skeletal tissues, liver, breast, skin, and kidney, and different tumor cells.

Types of Animal Cell Culture

Cell culture can be classified as primary cell culture and cell lines based on the number of cell divisions. Cell lines can undergo finite or infinite cell divisions.

A. Primary cell culture

This is the cell culture obtained straight from the host tissue cells. The cells dissociated from the parental tissue are grown on a suitable container, and the culture thus obtained is called primary cell culture. Such culture comprises mostly heterogeneous cells, and most cells divide only for a limited time. However, these cells are much similar to their parents.

Depending on their origin, primary cells grow either as an adherent monolayer or in a suspension.

Adherent cells

These cells are anchorage-dependent and propagate as a monolayer. These cells must be attached to a solid or semi-solid substrate for proliferation. These adhere to the culture vessel with the use of an extracellular matrix which is generally derived from tissues of organs that are immobile and embedded in a network of connective tissue. Fibroblasts and epithelial cells are of such types.

When the bottom of the culture vessel is covered with a continuous layer of cells, usually one cell in thickness, these are known as monolayer cultures. As single layers, such cells can be transferred directly to a coverslip to examine under a microscope. (continuous cell lines, covered later in this article also predominantly grow as adherent monolayers.)

Suspension cells

Suspension cells do not attach to the surface of the culture vessels. These cells are also called anchorage-independent or non-adherent cells which can be grown floating in the culture medium. Hematopoietic stem cells (derived from blood, spleen, and bone marrow) and tumor cells can be grown in suspension. These cells grow much faster, do not require the frequent replacement of the medium, and can be easily maintained. These are of homogeneous types and enzyme treatment is not required for the dissociation of cells; similarly, these cultures have a short lag period.

Confluent culture and the necessity of sub-culture

After the cells are isolated from the tissue and proliferated under the appropriate conditions, they occupy all of the available substrates i.e. reach confluence. For a few days, it can become too crowded for their container, which can be detrimental to their growth, generally leading to cell death if left for a long time. The cells thus have to be subculture i.e. a portion of cells is transferred to a new vessel with a fresh growth medium which provides more space and nutrients for the continual growth of cells. Hence subculture keeps cells healthy and in a growing state.

A passage number refers to how often a cell line has been sub-cultured. In contrast with the population doubling level, the specific number of cells involved is irrelevant. It gives a general indication of how old the cells may be for various assays.

B. Secondary cell culture and cell line

When a primary culture is sub-cultured, it is known as secondary culture, cell line, or sub-clone. The process involves removing the growth media and disassociating the adhered cells (usually enzymatically).

Sub-culturing primary cells into different divisions lead to the generation of cell lines. During the passage, cells with the highest growth capacity predominate, resulting in a degree of genotypic and phenotypic uniformity in the population. However, as they are sub-cultured serially, they become different from the original cell.

Based on the life span of culture, the cell lines are categorized into two types

Finite cell lines

The cell lines that go through a limited number of cell divisions with a limited life span are known as finite cell lines. The cells pass several times and then lose their ability to proliferate, a genetically determined event known as senescence. Cell lines derived from primary cultures of normal cells are finite cell lines.

Continuous cell lines

When a finite cell line undergoes transformation and acquires the ability to divide indefinitely, it becomes a continuous cell line. Such transformation or mutation can occur spontaneously, chemically, or virally induced or from the establishment of cell cultures from malignant tissue. Cell cultures prepared in this way can be sub-cultured and grown indefinitely as permanent cell lines and are immortal.

These cells are less adherent, fast-growing, less fastidious in their nutritional requirements, able to grow up to higher cell density, and different in phenotypes from the original tissue. Such cells grow more in suspension. They also tend to grow on top of each other in multilayers on culture-vessel surfaces.

Common cell lines

Human cell lines:

1. MCF-7 (breast cancer)
2. HL 60 (leukemia)
3. HeLa (human cervical cancer cells)

Primates cell lines: Vero (African green monkey kidney epithelial cells)

Cell strain

Lineage of cells originating from the primary culture is called strain. These are either derived from a primary culture or a cell line by the positive selection or cloning of cells having specific properties or characteristics. A cell strain often acquires additional genetic changes after initiating the parent line.

Figure: Fig: Animal Cell Culture

Methods

Growth Requirements

The culture media used for cell cultures are generally quite complex, and culture condition widely varies for each cell type. However, media generally include amino acids, vitamins, salts (maintain osmotic pressure), glucose, a bicarbonate buffer system (maintains a pH between 7.2 and 7.4), growth factors, hormones, O2 and CO2. To obtain the best growth, the addition of a small amount of blood serum is usually necessary, and several antibiotics, like penicillin and streptomycin, are added to prevent bacterial contamination.

Temperature varies with the type of host cell. Most mammalian cells are maintained at 37°C for optimal growth, while cells derived from cold-blooded animals tolerate a wider temperature range (15°C to 26°C). For sub-culturing, cells in the log (exponential) growth phase should be used, actively dividing cells will establish the new culture more reliably than those in stationary or declining phase.

Process to obtain primary cell culture

Primary cell cultures are prepared from fresh tissues. Pieces of tissues from the organ are removed aseptically, usually minced with a sharp sterile razor and dissociated by proteolytic enzymes (such as trypsin) that break apart the intercellular cement. The obtained cell suspension is washed with a physiological buffer (to remove the proteolytic enzymes used). The cell suspension is spread out on the bottom of a flat surface, such as a bottle or a Petri dish. This thin layer of cells adhering to the glass or plastic dish is overlaid with a suitable culture medium and is incubated at a suitable temperature.

Aseptic techniques

Bacterial infections, like Mycoplasma and fungal infections, commonly occur in cell culture, creating a problem to identify and eliminate. Thus, all cell culture work is done in a sterile environment with proper aseptic techniques. Work should be done in laminar flow with the constant unidirectional flow of HEPA filtered air over the work area. All the materials, solutions, and the whole atmosphere should be contamination-free.

Cryopreservation

If a surplus of cells is available from sub-culturing, they should be treated with the appropriate protective agent (e.g., DMSO or glycerol) and stored at temperatures below –130°C until needed.  This stores cell stocks and prevent the original cell from being lost due to unexpected equipment failure or biological contaminations. It also prevents finite cells from reaching senescence and minimizes the risks of changes in long-term cultures.

When thawing the cells, the frozen tube of cells is warmed quickly in warm water, rinsed with medium and serum, and then added into culture containers once suspended in the appropriate media.

Applications of Cell Line

A. Vaccines Production

One of the most essential uses of cell culture is in the research and production of vaccines. The ability to grow large amounts of virus in cell culture eventually led to the creation of the polio vaccine, and cells are still used today on a large scale to produce vaccines for many other diseases, like rabies, chickenpox, hepatitis B, and measles. In early times, researchers had to use live animals to grow poliovirus, but due to the development of cell culture techniques, they were able to achieve much greater control over virus production and on a much larger scale which eventually develop vaccines and various treatments. However, continuous cell lines are not used in virus production for human vaccines as these are derived from malignant tissue or possess malignant characteristics.

B. Virus cultivation and study

Cell culture is widely used to propagate viruses as it is convenient, economical, and easy to handle compared to other animals. It is easy to observe cytopathic effects, select particular cells on which the virus grows, and study the infectious cycle. Cell lines are convenient for virus research because cell material is continuously available. Continuous cell lines have been extremely useful in cultivating many previously difficult or impossible to grow viruses.

C. Cellular and molecular biology

Cell culture is one of the major tools used in cellular and molecular biology, providing excellent model systems for studying the normal physiology and biochemistry of cells (e.g., metabolic studies, aging), the effects of different toxic compounds on the cells, and mutagenesis and carcinogenesis. The major advantage of cell culture for any of these applications is the consistency and reproducibility of results obtained from a batch of clonal cells.

D. In Cancer Research

Normal cells can be transformed into cancer cells by methods including radiation, chemicals, and viruses. These cells can then be used to study cancer more closely and to test potential new treatments.

E. Gene therapy

Cells with a non-functional gene can be replaced by cells with functional genes, for which the cell culture technique is used.

F. Immunological studies

Cell culture techniques are used to know the working mechanism of various immune cells, cytokines, lymphoid cells, and the interaction between disease-causing agents and the host cells.

G. Others

Cell lines are also used in in-vitro fertilization (IVF) technology, recombinant protein, and drug selection and improvement.

How to Remember

Three types of culture in a progression from fresh to immortal. Primary cell culture is freshly harvested tissue — closest to the original organ, most physiologically faithful, but finite and variable batch to batch. Cell lines derived from it (secondary culture) have been passaged and selected, giving more uniformity. Within cell lines, finite lines eventually senesce; continuous cell lines have undergone transformation and grow indefinitely. Picture a spectrum: "most like the original body" at one end (primary cells), "furthest from it" at the other (continuous cell lines) — with biological realism and practical convenience trading off against each other as you move along the spectrum.

Adherent cells need a surface; suspension cells float — and this reflects where they came from in the body. Cells from solid organs (fibroblasts, epithelial cells, liver, kidney) are adherent in culture because they're anchored to connective tissue matrices in vivo. Cells from blood, lymph, and bone marrow (hematopoietic cells, lymphocytes, some tumour cells) float in vivo and do so in culture too. When you know where a cell came from, you can predict how it will grow in a flask.

Cryopreservation is insurance, and the cryoprotectant is the reason it works. Without DMSO or glycerol, freezing cells forms intracellular ice crystals that puncture cell membranes and kill the cells — exactly the disaster cryopreservation is designed to prevent. The agent enters the cell and displaces water, reducing ice crystal formation during slow controlled cooling to −130°C. Thaw quickly in warm water to reverse the process before crystal formation can occur on the way back up.

Continuous cell lines cannot make vaccines — because their transformation is why they're useful, and also why they're dangerous in vaccines. This same transformation that made them immortal and easy to maintain also made their DNA aneuploid and potentially oncogenic. Putting that DNA into a vaccine injected into healthy people is precisely what regulatory agencies prohibit. This is stated directly in the Applications section of this article, the "why" of the rule is the cell biology you've just read.

Key Exam Facts Table

Where Students Get Confused

"Secondary culture is a different type of culture, distinct from primary." It is not a distinct type — it is simply what primary culture becomes after its first sub-culturing passage. Primary culture passaged once = secondary culture. The terms describe stages in the same continuous cultivation process.

"Continuous cell lines are just faster-growing finite cell lines." They are biologically different, not just quantitatively faster. Finite cell lines retain normal genetic regulation and eventually senesce. Continuous cell lines have undergone transformation — a change in gene regulation that uncouples growth from normal cell-cycle controls, produces an aneuploid karyotype, and gives them indefinite replicative capacity. This is a qualitative biological change, not a speed difference.

"Suspension cultures are better because they grow faster and don't need enzymatic treatment." Neither culture type is inherently better — appropriateness depends on the cell's biology. Fibroblasts and epithelial cells are naturally adherent and will not grow properly in suspension; hematopoietic and many tumour cells are naturally non-adherent and cannot attach to a monolayer. Forcing the wrong culture format onto a cell type will produce poor or failed cultures.

"Cell line and cell strain are the same thing." They are not. A cell line is any culture derived from primary culture by sub-culturing. A cell strain is a subpopulation selected from a cell line or primary culture by cloning or positive selection for a specific property (a particular surface marker, a specific growth characteristic). All cell strains are derived from cell lines, but not all cell lines are strains — a strain has been specifically selected for defined characteristics.

References

1. Freshney, R. I. (2015). Culture of Animal Cells: A Manual of Basic Technique and Specialized Applications (7th ed.). Wiley-Blackwell.
2. Kuby, J., Kindt, T. J., Goldsby, R. A., & Osborne, B. A. (2006). Kuby Immunology (7th ed.). W. H. Freeman.
3. Pelczar, M. J., Chan, E. C. S., & Krieg, N. R. (1993). Microbiology: Concepts and Applications. McGraw-Hill.
4. Skloot, R. (2010). The Immortal Life of Henrietta Lacks. Crown Publishers.